CN107432875A - It is a kind of to be used to treat functional motor ability plastid of lung neoplasm and preparation method and application - Google Patents

It is a kind of to be used to treat functional motor ability plastid of lung neoplasm and preparation method and application Download PDF

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CN107432875A
CN107432875A CN201710790198.1A CN201710790198A CN107432875A CN 107432875 A CN107432875 A CN 107432875A CN 201710790198 A CN201710790198 A CN 201710790198A CN 107432875 A CN107432875 A CN 107432875A
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egcg
liposome
cell
liposomes
hpdl
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应雪
李霞
闫荷露
何兰玉
唐辉
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Shihezi University
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    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids

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Abstract

The invention discloses a kind of functional motor ability plastid for being used to treat lung neoplasm and preparation method and application.The target liposomes are prepared by lecithin, cholesterol, DSPE polyethylene glycol and DSPE polyethylene glycol folic acid.DSPE PEG2000 FA modifications are arrived surface of liposome by the present invention, while have the function that targets neoplastic cells；Antineoplastic dinatin and inducing apoptosis of tumour cell agent EGCG are contained in liposome, obtain multi-functional targeting lung neoplasm stem cell compound liposome, medicine can be made to reach lesions position, two medicines joint kills tumour cell and induced tumor stem cell apoptosis, greatly improve chemotherapy effect, it is contemplated that can effectively prevent tumor recurrence.The present invention brings hope for thoroughly radical cure lung neoplasm, has important theory significance and clinical meaning.

Description

It is a kind of to be used to treat functional motor ability plastid of lung neoplasm and preparation method and application
Technical field
The present invention relates to a kind of functional motor ability plastid and preparation method and application, and in particular to one kind swells for treating lung Functional motor ability plastid of knurl and preparation method and application, belong to lung neoplasm drug research field.
Background technology
In recent years, the morbidity and mortality of tumour are substantially rising, and lung cancer is global incidence and case fatality rate highest Malignant tumour, wherein non-small cell lung cancer (NSCLC) accounts for 80%, and the number of annual lung cancer death is about 1,500,000.NSCLC Grade malignancy is higher, and easily recurs and shift, and more than 50% patient is passed through to relative late period with regard to oneself once being diagnosed.Face Bed treatment uses the complex treatment based on radiation and chemotherapy more, but effect is unsatisfactory.Radiotherapy is difficult to the lung for eradicating infiltration metastasis Cancer cell, chemotherapy non-selectivity so that medicine is while cancer cell is killed also to normal cell damage.Therefore, find More effective and safety treatment means turn into a focus of current lung cancer research.
The prognosis of lung neoplasm is poor, is also due to caused by the tolerance of the tumour after surgery excision and tumor recurrence.It is most of swollen Knurl with the different proliferation potentials cell mass different with tumor activity is migrated to all by forming.Form the foreign cell of tumor entity There is the cell subsets that part has self-renewing and differentiation characteristic, i.e. tumor stem cell (cancer stem in group Cells, CSCs).One of leading indicator of the sensitiveness of clinical investigation radiation and chemotherapy and surgical effect is that tumour is thin at present Born of the same parents' quantity reduces or the degree of tumor mass reduction.But theoretical according to tumor stem cell (CSCs), this does not represent tumor stem cell It is fully erased, if treatment after tumor stem cell continue to breed, tumour is possible to recur after alleviation.It is so if complete The tumor stem cell in tumor tissues is killed entirely, then lung neoplasm just has the possibility effected a radical cure.Therefore, controlled in the targeting of tumor disease In treatment, tumor stem cell has become the target spot of the drug design and screening that receive much concern.
The content of the invention
It is an object of the invention to provide functional motor ability plastid for treating lung neoplasm and preparation method and application, this hair It is bright that surface of liposome is arrived into PEG-DSPE-folic acid (DSPE-PEG2- folic acid) modification, obtain one Kind target liposomes, may be used as pharmaceutical carrier and contain medicine, such as contain dinatin (HPDL, Hispidulin) and EGCG (Epigallo-catechin gallate (EGCG)) obtains pharmaceutical carrier, and two medicines are shared while antitumor action is improved, and strengthen it To tumour cell and its apoptosis-promoting effect of stem cell, possibility is provided to kill tumour cell and its stem cell, so as to prevent controlling Cause tumor recurrence because stem cell remains after treatment.
Target liposomes provided by the present invention, by lecithin, cholesterol, DSPE-poly- second two Alcohol and PEG-DSPE-folic acid (DSPE-PEG-FA) are prepared.
In above-mentioned target liposomes, the molfraction of each raw material can be：
The molfraction of each raw material is：
60~65 parts of the lecithin；
5~10 parts of the cholesterol；
1~5 part of the PEG-DSPE；
0.5~5 part of the PEG-DSPE-folic acid；
The molfraction of each raw material concretely it is following 1) or 2)：
1)
60~65 parts of the lecithin；
5~10 parts of the cholesterol；
1 part of the PEG-DSPE；
0.5~5 part of the PEG-DSPE-folic acid；
2)
63.86 parts of the lecithin；
9.83 parts of the cholesterol；
1 part of the PEG-DSPE；
0.83 part of the PEG-DSPE-folic acid.
The PEG-DSPE concretely DSPE-poly- second Glycol 2000 (DSPE-PEG2000)；
PEG-DSPE-the folic acid concretely DSPE-poly- Ethylene glycol 2000- folic acid (DSPE-PEG-FA).
Invention further provides the preparation method of the target liposomes, comprise the following steps：The lecithin, institute State cholesterol, the PEG-DSPE and the DSPE-poly- second two Alcohol-folic acid obtains the target liposomes by film dispersion method.
Pharmaceutical carrier using the target liposomes as active component falls within protection scope of the present invention.
The present invention also provides a kind of drug-loaded liposome, and it contains medicine by the target liposomes and obtained.
In the drug-loaded liposome, the medicine concretely dinatin and/or epigallocatechin nutgall Acid esters, dinatin are a kind of flavone compounds being present in Xinjiang Saussurea involucrate；Epigallo-catechin gallate (EGCG) It is a kind of composition extracted from Chinese green tea, it is the main activity and water-soluble components of green tea, is that content is most in catechin High component.
The medicine fat ratio of the drug-loaded liposome can be 1：10~30；
When the medicine is the dinatin, the medicine fat ratio can be 1：20；When the medicine is that the table does not have During infanticide catechin and gallate, the medicine fat ratio can be 1：10；When the medicine is the dinatin and the table During nutgall catechin gallic acid ester, the medicine fat ratio can be 1：6.7.
Quality and the lecithin in the target liposomes and the courage of the medicine fat than referring to the medicine The ratio of the quality sum of sterol.
Drug-loaded liposome provided by the invention can be used for treating lung neoplasm, concretely non-small cell lung cancer, more preferably lung Gland cancer.
Drug-loaded liposome provided by the invention can suppress lung tumor cell and/or lung neoplasm stem cells hyperplasia, promote lung Tumour cell and/or lung neoplasm stem cell apoptosis and enhancing lung tumor cell and/or lung neoplasm stem cell are to ingestion of medicines energy Power, the lung tumor cell are lung adenocarcinoma cell, concretely A549 cells, and the lung neoplasm stem cell can be that adenocarcinoma of lung is dry thin Born of the same parents, concretely A549 stem cells.
The present invention is ligand modified on surface of liposome by lung cancer target function of folic acid using dinatin as medicine, Epigallo-catechin gallate (EGCG) is that apoptosis-induced dose of lung neoplasm stem cell is contained in liposome, the brand-new tool of structure There is the drug-loaded liposome of lung cancer target function, medicine can be made to reach lesions position, targeting is concentrated on lung neoplasm Induced lung tumor Cell and its apoptosis of stem cell, greatly improve chemotherapy effect, effectively prevent tumor recurrence, are brought for thoroughly radical cure lung neoplasm Wish that there is important theory significance and clinical meaning.
Brief description of the drawings
Fig. 1 is the drug release patterns of drug-loaded liposome of the present invention (FA-HPDL-EGCG- liposomes), wherein, Fig. 1 (A) What is represented is in HPDL, HPDL+EGCG, HPDL- liposome, HPDL-EGCG- liposomes and FA-HPDL-EGCG- liposomes Dinatin drug release patterns, what Fig. 1 (B) was represented is EGCG, HPDL+EGCG, HPDL- liposome, HPDL-EGCG- fat EGGC drug release patterns in plastid and FA-HPDL-EGCG- liposomes.
Fig. 2 is Lung Adenocarcinoma A 549 Cell (Fig. 2 (A)) and tumor spheres (Fig. 2 (B)) form.
Fig. 3 is adenocarcinoma of lung A549 stem cell qualification results, and wherein Fig. 3 (A) represents CD133+Mark blank group, Fig. 3 (B) table Show CD133+Labelling experiment group；Fig. 3 (C) represents ALDH1+Mark blank group；Fig. 3 (D) represents ALDH1+Labelling experiment group.
Fig. 4 is inhibitory action of the different drug-loaded liposomes to Lung Adenocarcinoma A 549 Cell.
Fig. 5 is apoptotic effect of the different drug-loaded liposomes to A549 cells, wherein, Fig. 5 (A) represents free HPDL to lung gland Cancer A549 apoptotic effect, Fig. 5 (B) represent apoptotic effects of the free EGCG to adenocarcinoma of lung A549, and Fig. 5 (C) represents HPDL- lipids Body is to adenocarcinoma of lung A549 apoptotic effect, and Fig. 5 (D) expression EGCG- liposomes are to adenocarcinoma of lung A549 apoptotic effect, Fig. 5 (E) table Show apoptotic effects of the free HPDL+EGCG to adenocarcinoma of lung A549, Fig. 5 (F) represents HPDL-EGCG- liposomes to adenocarcinoma of lung A549 Apoptotic effect, Fig. 5 (G) represents that FA-HPDL-EGCG- liposomes represent blank to adenocarcinoma of lung A549 apoptotic effect, Fig. 5 (H) Apoptotic effect of the liposome to adenocarcinoma of lung A549.
Fig. 6 is that laser confocal detects distribution behavior of the cell to different drug-loaded liposomes in the cell, wherein, Fig. 6 (A), the fluorescence that Fig. 6 (B), Fig. 6 (C) and Fig. 6 (D) are represented respectively is medicine dinatin, PI, Hoechst33258 and above The superposition of three kinds of fluorograms.
Fig. 7 is intake ability of the Lung Adenocarcinoma A 549 Cell to different drug-loaded liposomes.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Reagent and instrument used in following embodiments is as follows：
1st, experiment reagent and cell
Egg yolk lecithin (EPC) is purchased from Germany Lipoid company, ShangHai local agent, China； Cholesterol (cholesterol) is purchased from Beijing bispin microbiological culture media products factory；Polyethylene glycol-distearoylphosphatidyl ethanol Amine (DSPE-PEG2000) is purchased from NOF Corp (Japanese NOF companies)；Dinatin is purchased from Shanghai with field biology Science and Technology Ltd.) EGCG is purchased from Chengdu Zhi Biaohuachun Bioisystech Co., Ltd；Ammonium sulfate ((NH4)2SO4), analysis is pure, purchase From Tianjin good fortune morning chemical reagent factory；PEG-DSPE 2000- folic acid is purchased from U.S. NANOCS public affairs Department；Chitosan is purchased from Shanghai Blue Season Technology Development Co., Ltd；Atoleine is purchased from Tianjin Yong Sheng Fine Chemical Co., Ltd； Span-80 is purchased from Beijing Suo Laibao Science and Technology Ltd；Tween-80 is purchased from Tianjin and recovers the research institute that becomes more meticulous；The high sugar of DMEM Culture medium, DMEM/F12 high glucose mediums, 0.25% pancreatin are purchased from Gibico companies；Hyclone is purchased from Hangzhou Chinese holly biology Science and Technology Ltd.；The dual anti-reagent of Pen .- Strep (100 ×) is purchased from Beijing Suo Laibao bio tech ltd；People's base This fibroblast growth factor (bFGF), hEGF (EGF) are purchased from PeproTech companies of the U.S.；B27 is purchased from U.S. Gibco companies of state；Sulforhodamine B albumen (SRB) is purchased from Sigma Co., USA.Lung Adenocarcinoma A 549 Cell is purchased from Chinese science Shanghai cell institute of institute.
2nd, laboratory apparatus
Poly- carbon ester filter (aperture 400,200nm) is purchased from Millipore (Bedford, MA, USA)；Waters e 2695 high performance liquid chromatographs (waters companies of the U.S.)；(Japanese Shimadzu is public for UV-2401PC types ultraviolet-visible spectrophotometer Department)；Malvern particle size analyzer (Malvern instrument company of Britain)；SartoriusBP211D electronic balances (German Sai Duolisi Group)；Ultrasonic cleaner (day roc New Electronic Techniques Beijing Co., Ltd)；EYELA-1000S types Rotary Evaporators (Japan EYELA Tokyo Physico-chemical Apparatus Co., Ltd.)；Vacuum drying chamber (PZE-6030, vacuum<133Pa, the upper grand experimental facilities of Nereid Co., Ltd)；ESEM (JSM-6490LV, JEOL)；(the new sesame biotechnology share in Ningbo has JY92 cells Ultrasonic Cell Disruptor Limit company)；Constant temperature oscillator (Medical Instruments factory of Jintan City of Jiangsu Province)；Bag filter (molecular cut off 8000-12000)；Lipid Body squeezer (German Sai Duolisi groups)；(Thermo science and technology is limited for flow cytometer (U.S. company BD) CO2 constant incubators Company, the U.S.)；Enzyme-labeled immunity analyzer (Shanghai Gene Tech. Company Limited).
The preparation of embodiment 1, liposome
First, the preparation of blank liposome
1st, the preparation of blank liposome
By lecithin, cholesterol and DSPE-PEG2000 in molar ratio 63.86：9.83：In methyl alcohol, rotation is steamed for 1 dissolving Hair removes methanol, 35 DEG C in eggplant-shape bottle, 40rpm rotary evaporation film forming, adds 250mM ammonium sulfate 10mL, first surpasses in a water bath Sound 5min, being then transferred into serum bottle in JY92 type ultrasonic cell disruptors further ultrasound, (the ultrasound works time is 10s, intermittent time 30s, all times 10min, protection temperature are 35 DEG C), reuse liposome squeezer mistake respectively Each 3 times of 400nm and 200nm polycarbon resin films, liposome foreign minister's liquid is changed with PBS liquid (pH=7.4) using dialysis, obtains blank Liposome.
2nd, the preparation of the blank liposome of PEG-DSPE 2000- modified with folic acid
By lecithin, cholesterol, DSPE-PEG2000 and DSPE-PEG-FA in molar ratio 63.86：9.83：1：0.83 is molten In methyl alcohol, rotary evaporation removes methanol to solution, 35 DEG C in eggplant-shape bottle, 40rpm rotary evaporation film forming, adds 250mM ammonium sulfate 10mL, first 5min ultrasonic in a water bath, is then transferred into serum bottle and further surpasses in JY92 type ultrasonic cell disruptors Sound (the ultrasound works time is 10s, intermittent time 30s, all times 10min, and protection temperature is 35 DEG C), reuses lipid Body squeezer crosses each 3 times of 400nm and 200nm polycarbon resin films respectively, and liposome is changed with PBS liquid (pH=7.4) using dialysis Foreign minister's liquid, obtain targetting blank liposome.
2nd, the preparation of drug-loaded liposome
Quality of the medicine fat than=dinatin and/or EGCG：The quality sum of lecithin and cholesterol in liposome.
By dinatin with 1：In methyl alcohol, operating method is same for the dissolving such as 20 medicine fat ratio and lecithin, cholesterol In step 1, obtain being loaded with the liposome (HPDL- liposomes) of dinatin.By EGCG with 1：10 medicine fat ratio and lecithin Fat, cholesterol etc. are dissolved in methanol and chloroform, the step 1 during operating method is same, obtain carrying EGCG liposome (EGCG- liposomes).By EGCG and HPDL with 1：6.7 medicine fat ratio and lecithin, cholesterol etc. are dissolved in methanol and three chloromethanes In alkane, the step 1 during operating method is same, obtain carrying dinatin and EGCG liposome (HPDL-EGCG- liposomes).
Dinatin-EGCG the liposomes that the above method obtains, obtain DSPE-PEG-FA according to the step 2 in one and repair The dinatin of decorations and EGCG liposome (FA-HPDL-EGCG- liposomes).
The performance measurement of embodiment 2, liposome
First, the measure of envelop rate
Non-encapsulated dinatin and EGGC, high effective liquid chromatography for measuring content are removed using dialysis.Take appropriate Dinatin liposome, EGCG liposomes, FA-HPDL-EGCG liposomes add acetonitrile to clear up before dialysis, determine dinatin And EGCG content；The another dinatin liposome taken after dialysing, EGCG liposomes, FA-HPDL-EGCG liposomes add acetonitrile Resolution, the EGCG contained in liposome content is determined, entrapment efficiency is calculated according to formula (1)：
Envelop rate %=WBag/WAlways× 100% (1)
Wherein WAlwaysFor total dose；WBagRepresent the dose of liposome entrapment.
In prepared drug-loaded liposome, the envelop rate of dinatin is reachable up to 86%, EGCG envelop rate 75%.
2nd, particle diameter and potential measurement
Respectively by blank liposome freshly prepared 1mL, HPDL- liposomes, EGCG- liposomes and HPDL-EGCG- liposomes Diluted with FA-HPDL-EGCG- liposomes with PBS, use Nano Series Zen 4003Zeta Sizer (Malvern Instruments, Ltd, UK) measure liposome particle diameter, polydispersity coefficient and Zeta potential.It is the particle diameter of different liposome, more The measurement result of the coefficient of dispersion (PDI) and zeta current potentials is shown in Table 1.Liposomal particle size is evenly distributed, particle size 90nm~ Between 120nm.Each drug-loaded liposome all slightly negative electricity, illustrate there is appropriate electrostatic repulsion forces between liposome vesicle, store Stability is more preferable, is not susceptible to coagulation.
The different liposome particle diameter of table 1 and Zeta potential (n=3)
3rd, the release of drug-loaded liposome
Precision measures the free HPFL of 1.00mL, free EGCG, HPDL- liposome, EGCG- liposomes, HPDL- respectively EGCG- liposomes, FA-HPDL-EGCG liposome turbid liquors are transferred in the bag filter through processing, fasten bag filter both ends, suspension In the conical flask for filling 25 times of dissolution mediums, it is placed in constant temperature oscillator, the constant temperature oscillation 48h in 37 ± 0.5 DEG C, is setting Sample time sampling, and fill into isometric dialysis medium immediately, cross 0.45 μm of miillpore filter, μ L, the HPLC analyses of sample introduction 20 are surveyed It is fixed.HPDL and EGCG release percentage is calculated according to formula (2)：
F (%)=(Ct/CAlways) × 100% (2)
Ct/CAlwaysFor the ratio between each time point measured concentration and concentration in the dissolution medium that is calculated by labelled amount, thus ask The release percentage F (%) of different time is obtained, draws release profiles.
From Fig. 1 (A), the free medicine rate of release of dinatin is very fast, HPDL- when reaching 88%, 48h in 48h Dinatin release rate is in liposome, EGCG- liposomes, HPDL-EGCG- liposomes and FA-HPDL-EGCG- liposomes 30% or so, release is slow, no phenomenon of burst release.From Fig. 1 (B), the free medicine rate of release of EGCG is very fast, reaches in 48h EGCG release rates are in EGCG- liposomes, HPDL-EGCG- liposomes and FA-HPDL-EGCG- liposomes when more than 86%, 48h 30% or so, release is slow, no phenomenon of burst release.The above results show, steady when dinatin and EGCG are using liposome as carrier Qualitative good, Target liposomes have slow releasing function.
The inhibitory action research to Lung Adenocarcinoma A 549 Cell in vitro of embodiment 3, drug-loaded liposome
First, condition of culture
1st, Lung Adenocarcinoma A 549 Cell condition of culture
Lung Adenocarcinoma A 549 Cell is incubated at containing 10% hyclone, 100U/mL penicillin and 100 μ g/mL streptomysins In DMEMF12 high glucose mediums, 37 DEG C, 5%CO2Saturated humidity incubator is incubated, Lung Adenocarcinoma A 549 Cell adherent growth, cell Form is in fusiformis, and (shown in Fig. 2 (A)), growth period cell of taking the logarithm is tested.
2nd, adenocarcinoma of lung A549 stem cell cultivation conditions
The A549 cells in exponential phase are taken, with PBS, the digestion of 0.25% pancreatin, blow and beat into single cell suspension, Serum free medium (containing DMEM/F12, bFGF, EGF, B27) is resuspended in, cell propagation can form suspension growth after 4~5 days Tumor spheres, spherical in shape, cell is completely embedded, shown in form such as Fig. 2 (B).The propagating method of stem cell sphere is specially： The tumor spheres of suspension growth are transferred in test tube together with culture medium, 5min is centrifuged with 1000r/min, abandoned Clearly, cell is resuspended in serum free medium, blows and beats repeatedly, resolves into cell mass unicellular, by 1：2 or 1：3 ratio inoculation In 25cm2Floor space blake bottle, addition serum free medium continue in 37 DEG C, 5%CO to 6mL/ bottles2In saturated humidity incubator Culture, the A549 stem cell spheres of culture 14 days are taken to be tested.
3rd, the discriminating of stem cell
Due to CD133+And ALDH1+In the high expression of stem cell surface, therefore for identifying A549 stem cells.
CD133 labeled stem cells are tested：
Former stem cell liquid is poured into centrifuge tube, 5min is centrifuged, abandoning supernatant, PBS (containing 0.5%BSA) washings, uses 0.25% pancreatin is digested, and nutrient solution terminates digestion and centrifuged again, after being washed after abandoning supernatant using PBS, is added certain Cell culture fluid so that the concentration of cell be 1 × 106.After preparing cell suspension, each 100 μ L of two solencyte liquid are taken respectively, One pipe is control group, and a pipe is experimental group.Blank control group adds 5 μ LPBS, and experimental group adds 5 μ LCD133 antibody, and lucifuge is incubated 30min is educated, is centrifuged again, is washed after centrifugation using PBS.300 μ LPBS are resuspended, upper machine testing.
4th, Aldefluor kits identification stem cell experiment
Kit is put into (15-25 DEG C) at room temperature using preceding, 25 μ L are added in the bottle of Aldefluor dry powder DMSO, at room temperature blending incubation 1 minute (Aldefluor dry powder is Chinese red, becomes yellow green when adding DMSO).Then add 25 μ L 2N HCl, mix, be incubated 15 minutes at room temperature.360 μ L Aldefluor buffer solutions are added in reagent bottle fully to mix After dispense, -20 DEG C preservation.
Cell after centrifuging is taken in addition, and Aldefluor buffer are resuspended, and adjustment cell number is 1 × 106/ mL, mark A549 Groups of cells and suspension cell ball group, and labelling experiment group and control group respectively.1mL cells are separately added into experimental group EP pipes to hang Liquid, 5uL DEAB inhibitor is added in control group EP pipes, covering the lids of EP pipes rapidly, (DEAB is dissolved in 95% alcohol In, should prevent from volatilizing).
The Aldefluor of 5 μ L activation is added in experimental group, after mixing, transferase 10 .5mL mixed liquors are into control group immediately (test tube for adding DEAB).Because ALDH enzymatic reactions are rapid, after Aldefluor reagents are added should immediately transferase 10 .5mL to plus In DEAB control EP pipes.
Experimental group and control group EP are managed, after 37 DEG C are incubated 45 minutes, 1000r/m, centrifuge 5min.Abandoning supernatant, Cell is resuspended in Aldefluor buffer solutions, after be placed on ice or 4 DEG C.Treat flow cytometry analysis ALDH1high cell proportions.
From the figure 3, it may be seen that compared in Isotype control, CD133 in adenocarcinoma of lung A549 stem cells+Expression quantity is 98.0%, ALDH1+ Expression quantity be 71.4%.CD133 as can be seen from the results+And ALDH1+In the adenocarcinoma of lung A549 stem cell spheres cultivated Height expression, illustrate that above-mentioned condition of culture is adapted to growth and the propagation of stem cell.
2nd, the drug-loaded liposome of various concentrations is prepared
Prepare free dinatin, free EGCG, free HPDL-EGCG, HPDL- liposome, the EGCG- of series concentration Liposome, HPDL-EGCG- liposomes and FA-HPDL-EGCG- liposomes, 10 μ L said medicine solution are taken to be separately added into cell training Support in hole, the drug concentration gradient for making each experimental group final is：0、0.5、1、5、10、20、30、40μM.
3rd, antiproliferative effect of the drug-loaded liposome to Lung Adenocarcinoma A 549 Cell
The take the logarithm μ L/ holes of 3000/hole Lung Adenocarcinoma A 549 Cell 190 in growth period are inoculated in 96 orifice plates, 37 DEG C, and 5% CO2Under the conditions of cultivate 24h；The free dinatin, free EGCG, free HPDL-EGCG, HPDL- of series concentration will be prepared Liposome, EGCG- liposomes, HPDL-EGCG- liposomes and FA-HPDL-EGCG- liposomes respectively take 10 μ L to be separately added into cell In culture hole, each concentration sets four multiple holes.Continue to cultivate 24h after administration, obtained through SRB methods per hole trap OD values (540nm), inhibitory action of the different drug-loaded liposomes to A549 cells is calculated, as shown in Figure 4.It is figure 4, it is seen that free HPDL is weaker to the inhibitory action of A549 cells, but with after EGCG combinations, the growth inhibition work to A549 cells can be remarkably reinforced With；FA-HPDL-EGCG- liposomes are better than other drug-loaded liposome groups to A549 antiproliferative effect.
Embodiment 4, drug-loaded liposome are studied the apoptotic effect of Lung Adenocarcinoma A 549 Cell in vitro
Take the logarithm growth period Lung Adenocarcinoma A 549 Cell with 2 × 105Individual/hole is inoculated in 6 orifice plates, 37 DEG C, 5%CO2Culture Cultivated in case.After cell growth 24h, add free dinatin, free EGCG, free HPDL-EGCG, HPDL- liposome, EGCG- liposomes, HPDL-EGCG- liposomes and FA-HPDL-EGCG- liposomes final concentration of 10 μM of EGCG (HPDL and), Each medicine sets three multiple holes.Put after continuing to cultivate 24h in incubator, rinsed 3 times with cold PBS, the pancreatin digestion without EDTA, Cell is washed with PBS twice, the PBS that 300 μ L flow back blows the sinking cell of centrifugation even after centrifugation (1000rpm centrifuges 3min). 500 μ L Binding Buffer, 5 μ L Annexin V-APC, 5 μ L 7-AAD is sequentially added afterwards, is mixed and is kept away after room temperature 10min is reacted under conditions of light, through flow cytomery.
Experimental result as shown in figure 5, free dinatin, free EGCG, free HPDL-EGCG, HPDL- liposome, EGCG- liposomes, HPDL-EGCG- liposomes and FA-HPDL-EGCG- liposomes are respectively to the apoptosis rate of A549 cells 9.5%th, 14.1%, 23.4%, 25.5%, 31.3%, 33.7% and 42.0%.The coarse wool artemisiifolia it can be seen from the above results Element itself has apoptosis-promoting effect to A549 cell lung gland cells, but effect is weaker, adds after EGCG and A549 cells are promoted to wither Die effect increase；Dinatin-EGCG by the use of liposome as carrier after the apoptosis-promoting effects of A549 cells is substantially increased.Through Statistical analysis, there is significant difference compared with other each groups, illustrate that its apoptosis-promoting effect is significantly stronger than other each groups.
Embodiment 5, Laser Scanning Confocal Microscope study distribution of the different drug-loaded liposomes in Lung Adenocarcinoma A 549 Cell
A549 cells are inoculated in 12 orifice plates, inoculum density is 2 × 104Individual/hole, after 24h, adherent growth, by FA- HPDL-EGCG-L, HPDL-EGCG-L, HPDL+EGCG, EGCG-L, HPDL-L and HPDL, EGGC are separately added into each hole (coarse wool Final concentration of 10 μM of ambrosin and EGGC), put in CO2gas incubator, 37 DEG C are cultivated 2 hours, are rinsed successively with cold PBS Three times, after the fixation of 4% paraformaldehyde 10min, Hoechst33258 nuclear targetings 5min, PI dyeing 5min.Laser co-focusing Microscope excites dinatin with the laser beam of 480nm wavelength, and Hoechst33258 is excited with the laser beam of 346nm wavelength, Then graphical analysis is carried out with Aim Image Examiner softwares.
Laser Scanning Confocal Microscope Fig. 6 shows that each preparation of dinatin is mainly distributed on after A549 lung adenocarcinoma cell endocytosis In nucleus, as illustrated, compared with other each groups, the red fluorescence of A549 cells after targeting FA-HPDL-EGGC-L effects Intensity is most strong, shows that it is most in the intracellular abundances of A549, illustrates that target liposomes can strengthen A549 cells in medicine Gulp down effect.
The intake experiment of embodiment 6, Lung Adenocarcinoma A 549 Cell to different drug-loaded liposomes
An importance for particulate delivery system interior evaluating is the absorption and transhipment for studying it in vivo at present Situation, more common method are to transport behavior inside tracer delivery system after carrying out fluorescence labeling.Coumarin 6 (Coumarin- 6) it is a kind of laser conversion ratio is higher, performance comparision is stable fat-soluble laser dye.This experiment is prepared using film dispersion method The liposome of model drug dinatin and fluorescent marker coumarin 6 is contained, studies the external intake situation of A549 cells.
A549 cells are taken, are seeded in after collecting centrifugation on 12 orifice plates containing slide, serum-containing media culture, 20,000/ Hole, in 37 DEG C of CO2gas incubators, 24h is incubated under conditions of relative humidity 95%.Be separately added into free dinatin, Free EGCG, free HPDL-EGCG, HPDL- liposome, EGCG- liposomes, HPDL-EGCG- liposomes and FA-HPDL- EGCG- liposomes.Put after continuing to cultivate 2h in incubator, after incubation, cell is washed three times with cold PBS, through 0.25% Collected by trypsinisation, then blown and beaten with PBS into cell suspension, through flow cytomery.As a result as shown in fig. 7, in A549 cells Intake experiment in, compared with free medicine and other each group drug-loaded liposomes, FA-HPDL-EGCG-- cumarins-liposome is shown Go out the intake ability of most strong A549 cells.
In summary, the multifunctional targeted liposome that the present invention is built, particle size is homogeneous, and insoluble drug release is stable；To lung Gland cancer A549 cells have very strong antiproliferative effect；Dramatically increase intake of the Lung Adenocarcinoma A 549 Cell to medicine；It can induce lung The apoptosis of gland cancer A549 cells；It can significantly improve the ability that liposome penetrates lung cancer, and then by drug delivery to tumor locus, kill Dead tumour cell, prevent tumor recurrence.

Claims (5)

  1. A kind of 1. target liposomes, it is characterised in that：The target liposomes are by lecithin, cholesterol, distearoylphosphatidyl Monoethanolamine-polyethylene glycol and PEG-DSPE-folic acid are prepared.
  2. 2. target liposomes according to claim 1, it is characterised in that：In the target liposomes, mole of each raw material Number is：
    The molfraction of each raw material is：
    60~65 parts of the lecithin；
    5~10 parts of the cholesterol；
    1~5 part of the PEG-DSPE；
    0.5~5 part of the PEG-DSPE-folic acid.
  3. 3. the preparation method of the target liposomes of claim 1 or 2, comprises the following steps：The lecithin, the courage are consolidated Alcohol, the PEG-DSPE and the PEG-DSPE-folic acid The target liposomes are obtained by film dispersion method.
  4. 4. a kind of pharmaceutical carrier, its active component is the target liposomes of claim 1 or 2.
  5. 5. a kind of drug-loaded liposome, contain medicine by the target liposomes of claim 1 or 2 and obtain.
CN201710790198.1A 2017-09-05 2017-09-05 It is a kind of to be used to treat functional motor ability plastid of lung neoplasm and preparation method and application Pending CN107432875A (en)

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Application publication date: 20171205